This Program Project of 26 years duration brings together investigators with a wide range of expertise to focus on gap unctions, which are intercellular channels comprised of connexins. The overall goal is to analyze physiological roles of gap unctions formed by connexins that are found in the nervous system. Molecular biology, culture of specific cell types and of brain slices, patch clamping, Northern and Western analysis, in situ hybridization, and site specific antibodies detected by confocal and electron microscopy provide a powerful combination for analysis of function. Communication by gap junctions can be reduced by antisense DNAs and by gene knockout through homologous recombination, and it can be increased by transfection with selected connexins including those not normally expressed. These approaches are highly specific and applicable both in vivo and in vitro , and gap junctions are being investigated in ways previously unforeseen. Project 1. Gap junctions and Schwann cells addresses the role of gap junctions between Schwann cells in normal, degenerating and regenerating nerve. Schwann cells express Cx32 when myelinating but change over to expressing Cx46 in culture. The role of these connexins in normal development and Wallerian degeneration following nerve lesions will be investigated using wild type and connexin knockout mice. The action of cytokines/growth factors on gap junction expression and how gap junctions affect the response to these factors will be determined. These studies are given particular relevance by the finding that the X-linked form of Charcot-Marie-Tooth disease can be caused by a loss-of-function mutation in Cx32. Project 2. Gap junctions and astrocytes addresses the role of gap junctions in propagation of signals between astrocytes and in their secretory and uptake processes. The regulation of expression of connexins and their role in gliosis will be examined. Cultured brain slices will be used to determine the involvement of astrocytic gap junctions in epilepsy and spreading depression. Mice in which the gene for the major astrocyte connexin, Cx43, is knocked out, will be an important tool in these studies. Project 3. Biophysics of nervous system connexins will use recombinant techniques to characterize the channels of gap junctions that the can form between these connexins and will seek to identify functionally significant domains of the molecules through sequence comparisons, domain swapping and site directed mutagenesis. Of particular interest are domains that mediate dependence on transjunctional voltage and affect permeation. A new preparation of isolated, functional hemichannels allows improved time resolution of responses to voltage and rapid solution changes that will simplify permeability measurements and clarify of the mode of action of the classical blocking agents, Ca2+ and H+. Cytology, Confocal Microscope and Antibody/Protein Cores provide cutting edge cytological techniques to provide the essential structural correlates to the physiological aspects of the Program. Together, these studies will increase our understanding of the many physiological and pathological processes in which gap junctions are involved, and findings of the Program ultimately will have implications for therapeutic intervention.